Ser217Cys mutation in the Ig II domain of FGFR3 in a Chinese family with autosomal dominant achondroplasia

Abstract

Achondroplasia (ACH) is the most common form of skeletal dysplasia characterized by disproportionately short stature, lumbar lordosis, relative macrocephaly and other skeletal anomalies resulting from a defect in the maturation of the chondrocytes in the growth plate of the cartilage. The combined frequency of the disease has been estimated to be 1 in 15 000 live births.1 ACH is inherited in autosomal dominant fashion with a complete penetrance, more than 80% of affected individuals have de novo mutations associated with increased paternal age. ACH is caused by mutations in FGFR3 gene, which contains 19 exons spanning 16.5 kb and encodes a membrane-spanning tyrosine kinase receptor of 806 amino acids.2 The fibroblast growth factor 3 is one member of the family of polypeptide growth factors involved in a variety of activities, including mitogenesis, angiogenesis and wound healing. FGFR3 contains three different parts: three extracellular immunoglobulin-like loops (Ig I-III), one single hydrophobic transmembrane domain and two cytoplasmic tyrosine kinase (TK) sub-domains TK1 and TK2 responsible for the catalytic activity.3 A predominant FGFR3 mutation, G to A transition at nucleotide 1138, has been found in more than 98% of the affected individuals. A second mutation described in about 2% of ACH patients is G to C transversion at the same codon. Both mutations resulted in the substitution of an arginine residue for a glycine at position 380 of the mature protein, which is in the transmembrane domain of FGFR3.4,5 A few of additional missense mutations have been reported as pathogenic cause. In the Chinese population, Ni et al5 studied 17 ACH cases and found the G380R was only responsible for 14 patients. In a Chinese family with ACH, T394S was identified as causative mutation.6 These results suggested that there are other mutations accounting for a few ACH patients. In this study, we analyzed a Chinese family with autosomal dominant achondroplasia. Using linkage analysis, we mapped the disease locus to chromosome 4p16.3, where the FGFR3 gene is located. Using direct DNA sequence analysis we identified a novel Ser217Cys mutation in exon 5 of FGFR3 that causes autosomal dominant achondroplasia. METHODS Study participants A three generation Chinese family with clinically diagnosed as ACH was enrolled in this study in Zhejiang, China (Fig. 1A). The proband (III-7) was a 36-year old man. He is 135 cm tall, with normal intelligence, good general health and can do some simple job. He has typical characteristic changes, including a disproportionately large head, large protruding forehead, flat nasal bridge, short maxilla, large mandible, thickened and protuberant lips, short trident hands, rhizomelic shortening of the long bones, neurologic dysfunction due to thoracolumbar spinal stenosis, scoliosis, lumbar hyperlordosis and short ribs. Embowed lower limbs cause the stagger.Fig. 1. A:: Pedigree of a Chinese family with autosomal dominant achondroplasia. B: In restriction fragment length polymorphism (RFLP) analysis, the wild type allele is represented by the 510 bp DNA band, and the mutant allele is shown as two bands (227 and 283 bp). RFLP analysis showed that the novel Ser217Cys mutation co-segregated with the affected individuals.Isolation of genomic DNA Informed consent was obtained from the participants in the ACH family. Genomic DNA was extracted from the peripheral blood with DNA isolation kit for mammalian blood (Promega, USA), according to the protocol of manufacturer. Linkage analysis Polymorphic microsatellite markers flanking the FGFR3 were selected from the ABI PRISM linkage mapping set-MD10 panel for linkage analysis. Genotyping was carried out using an ABI 3100 Genetic Analyzer, and allele-typing was performed using the GeneMapper 2.5 software (Applied Biosystems, Foster City, CA). DNA sequence analysis and mutation detection The whole coding region and exon-intron boundaries of FGFR3 were PCR-amplified. PCR primer pairs were designed from flanking intronic sequences. The PCR products were extracted using the QIAquick Gel Extraction Kit (Qiagen Inc., Valencia, CA), and sequenced with both forward and reverse primers. DNA sequence analysis was performed using the BigDye Terminator Cycle Sequencing v3.1 kit and on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, USA). Restriction fragment length polymorphism (RFLP) analysis The Ser217Cys mutation generated a novel BsaMI restriction enzyme site (GAATGCN) in exon 5 of the FGFR3 gene, we PCR-amplified exon 5 and flanking sequences from all the family members and 200 normal controls. Primers used were forward: 5′-AACACCGTCCGCTTCCGCT-3′; reverse: 5′-TGCGTCACTGTACACCTTGCA-3′. The PCR products were digested with BsaMI (Promega, Madison, USA) at 65°C for 4 hours, and separated on a 2.5% agarose gel. RESULTS We characterized a three generation Chinese family with ACH. Six family members (4 affected and 2 unaffected) participated in this study (Fig. 1A). To identify the causative mutation, the entire coding region and exon-intron boundaries of FGFR3 were sequenced, and found a single nucleotide change A to T at nucleotide 649 from the start codon ATG (NM_000142). The A to T change results in substitution of the serine residue at codon 217 by a cysteine residue (Figs. 2A and 2B). The Ser217 residue is highly conserved during evolution and is located in the extracellular domain, the second Ig-like domain (Fig. 2C).Fig. 2.: DNA sequencing in exon 5 of FGFR3 for proband III7 with mutation Ser217Cys (A, affected) and a normal family member (B, unaffected). The boxed codon 217 is a mutation (A to C), which results in the substitution of a serine residue by a cysteine residue. C: The alignment of amino acids of FGFR3 from Mus musculus, Bos taurus, Macaca mulatta to humans (Homo sapiens). The Ser217 residue is highly conserved during the evolution in the extracellular domain, the second Ig-like domain.Direct DNA sequence analysis of the members of the family showed that the Ser217Cys mutation co-segregated with all affected individuals, and was not present in unaffected family members. This mutation created a novel BsaMI restriction enzyme site (GAATGCN) in the mutant allele. RFLP analysis confirmed the results of DNA sequence analysis (Fig. 1B). The proband and his affected two sisters (III-2 and III-3) and one brother (III-5) carry the mutation allele (with bands of 227 and 283 bp). All other family members carried only the wild type allele (510 bp) (Fig. 1B). Further RFLP analysis did not detect the Ser217Cys mutation in 200 normal controls. These results suggested that the Ser217Cys mutation of FGFR3 is not a rare polymorphism, but a causative mutation for ACH in the Chinese family. DISCUSSION In this study, we investigated a Chinese family with autosomal dominant achondroplasia using linkage analysis and direct DNA sequence and identified a 649 A to T transversion in the FGFR3 gene, resulting in a novel Ser217Cys substitution. Ser217Cys was not present in other normal family members and 200 normal controls, suggests that this substitution is a pathogenic mutation causing ACH in the family. Mutations in the gene for human FGFR3 cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, ACH, thanatophoric dysplasia (TD),7 and hypochondroplasia (HCH),8 and recently another novel mutation in FGFR3 which causes camptodactyly, tall stature, and hearing loss (CATSHL) syndrome has been reported.9 More than 98% of the patients with achondroplasia from different ethnic groups carried the G380R mutation resulting from G to A transition at position 1138 in FGFR3 gene, which located in the transmembrance domain. In 2% of the patients, it has been shown that G to C transversion at the same position caused G380R mutation. In this study, neither of the two predominant mutations was detected, whereas a novel mutation Ser217Cys has been identified in the Chinese family. S217 located in the second Ig-like domain in the extracellular portion. To our knowledge, this is the first identified mutation causing ACH, which occurring in the Ig II loop of FGFR3. Very recently, another R200C mutation in this domain has been found responsible for HCH, a similar skeletal dysplasia.8 These results confirm that mutations outside the TM and TK domains and creating cysteine residues in the extracellular region can also cause ACH and severe HCH. It is elusive why different mutations of FGFR3 cause a variety of skeletal dysplasias. The novel Ser217Cys mutation has the similar character as most of TD mutations and the R200C mutation of HCH. They all create a novel cysteine residue and locate in the extracellular domain of the receptor;10 but ACH clinical phenotype is obviously milder than TD, while more severe than HCH. The resultant mutant FGFR3 proteins causing ACH, TD and HCH can constitutively activate the receptor, while the mutants of FGFR3 responsible for TD are more strongly activated than the mutation causing ACH and HCH. FGFR3 is a negative regulator of chondrocyte proliferation and differentiation in the growth plate. Mutations causing ACH and other related skeleton disorders activated the receptor and can have effect as gain-of function. The typical mutant G380R introduces a positively charged residue within the interior of the hydrophobic membrane may cause a change in receptor protein conformation crucial for the interaction of the receptor with intracellular components, or may have a specific block in the transmission of signal by the mutant receptor causing the lack of down-modulation. Compared with G380R, the novel Ser217Cys mutation in this study creates cysteine residue in the second Ig-like domain, which may allow disulfide bonds to form between the extracellular domains of mutant monomers, thus inducing constitutive activation of the homodimer receptor complex, but it remains to be further investigated whether Ser217Cys mutation like TDI and TDII can lead to ligand-independent constitutive phosphorylation, or like G380R has a specific defect in ligand-mediated receptor down-regulation.